Nuevos mecanismos moleculares de tolerancia a sequía y otros tipos de estrés abiótico en especies arbóreas de interés económico

Las masas forestales tienen una importancia colosal para nuestra sociedad y el conjunto de la biosfera. Estudios recientes a escala mundial indican que la sequía es el factor abiótico que más afecta a su crecimiento y supervivencia, seguida por las temperaturas extremas y la salinidad. Aunque comprender los mecanismos con que las especies arbóreas toleran estas formas de estrés tiene un interés aplicado evidente, dichos mecanismos se han estudiado mucho más en especies herbáceas modelo o de interés agronómico. Existen sin embargo diferencias notables entre ellas, como se demuestra en esta tesis y en otros trabajos recientes. Nuestro estudio se centra concretamente en la respuesta molecular del chopo –el sistema modelo forestal más desarrollado– al estrés abiótico, con particular énfasis en la sequía. Utilizando una estrategia proteómica y tratamientos controlados, hemos identificado componentes mayoritarios de dicha respuesta. Su participación en la misma se ha validado mediante análisis transcripcionales detallados utilizando tecnología qRT-PCR (PCR cuantitativa en tiempo real). Hemos identificado proteínas cuyo nexo funcional con mecanismos de tolerancia ya era conocido, como chaperonas moleculares sHSP o enzimas que atenúan el estrés oxidativo, pero también proteínas cuya relación funcional con el estrés es menos clara o incluso novedosa, como polifenol oxidasas (PPO), deshidrogenasas/reductasas de cadena corta (SDR), o bicupinas (BIC), entre otras. El cuerpo central de la tesis consiste en la caracterización detallada de una PPO inusual, cuya inducción por estrés hídrico se describe por vez primera. Estas enzimas están ampliamente distribuidas en plantas, si bien su número es muy variable de unas especies a otras. Algunas, como nogal, tienen un único gen, mientras que Arabidopsis no tiene ninguno. En la última versión del genoma de chopo hemos identificado un total de 12 miembros bona fide, corrigiendo trabajos previos, y hemos caracterizado su expresión individual ante diferentes situaciones de estrés controlado y tratamientos hormonales. La isoforma antedicha es el único miembro de la familia que responde claramente a la deshidratación. También responde a salinidad y a la mayor parte de tratamientos hormonales ensayados, pero no a daño mecánico o tratamientos con metil jasmonato. Esto la diferencia de enzimas homólogas presentes en otras especies de plantas, que se han relacionado experimentalmente con estrés biótico. Los patrones de acumulación de transcritos en árboles adultos son compatibles con un papel protector frente a la sequía. La integración de nuestros estudios funcionales y filogenéticos sugiere que la familia ha sufrido un proceso reciente de diversificación y neofuncionalización, siendo la protección frente a deshidratación su papel primigenio. Aunque se conoce la actividad bioquímica in vitro de este tipo de enzimas, sus sustratos naturales son esencialmente una incógnita. Mediante expresión heteróloga en Escherichia coli BL21(DE3) hemos detectado que la enzima de chopo es capaz de oxidar L-DOPA a dopaquinona, siendo menos activa frente a otros sustratos. Por otra parte, hemos demostrado su localización cloroplástica mediante transformación transitoria de protoplastos con fusiones a la proteína fluorescente YFP. Mediante la obtención de plantas transgénicas de A. thaliana hemos demostrado que la enzima de chopo aumenta considerablemente la tolerancia in vivo frente a la deshidratación y al estrés salino. El análisis fenotípico detallado de las líneas transgénicas, combinando múltiples metodologías, nos ha permitido sustanciar que la tolerancia tiene una base compleja. Esta incluye una mayor protección del sistema fotosintético, una capacidad antioxidante muy incrementada y la acumulación de solutos osmoprotectores como la prolina. Los análisis metabolómicos nos han permitido asociar la expresión de la proteína a la síntesis de un flavano no descrito previamente en A. thaliana, vinculando la enzima de chopo con la síntesis de fenilpropanoides. También hemos observado alteraciones en los niveles hormonales que podrían subyacer a efectos pleiotrópicos con interés aplicado, como un aumento consistente del tamaño de la planta o el acortamiento del ciclo de crecimiento. Además de aportar datos novedosos sobre la funcionalidad in vivo de esta familia de oxidasas, los resultados de esta tesis demuestran que los árboles son sistemas de estudio interesantes para caracterizar nuevas estrategias de tolerancia al estrés abiótico con potencial aplicado. ABSTRACT Forests masses have an extraordinary importance for our society and the biosphere. Recent worldwide studies indicate that drought is the abiotic factor that affects more their growing and survival, followed by extreme temperatures and salinity. The understanding of how the arboreal species tolerate the stress has an evident practical interest, but their mechanisms have been studied much more in herbaceous species or with agronomic interest. However, considerable differences exist between them, as this thesis and recent studies show. Our study is focused on the molecular response of the poplar –the more developed forestry model system- to abiotic stress, specifically focused in the drought. Using a proteomic strategy and controlled treatments, we have identified main components in such response. Its participation has been validated through transcriptional analysis using qRT-PCR technology. We have identified proteins whose functional connection with tolerance mechanisms were already known, as molecular chaperones sHSP or enzymes that attenuate the oxidative stress, but also some proteins whose functional relationship with the stress is less clear or even novel, as polifenol oxidases (PPO), short chain deshidrogenases/reductases (SDR), or bicupines (BIC), among others. The central body of the thesis consists of the detailed characterization of an unsual PPO, whose induction due to drought stress is first described. These enzymes are thoroughly distributed in plants, but their number of members is very variable among species. Some of them, as the walnut tree, have a single gene, while Arabidopsis has none. We have identified a total of 12 members in the last version of the poplar genome, correcting previous works, and have characterized their individual expression against different situations of controlled stress and hormone treatments. The aforementioned isoform is the only member of the family that responds clearly to the drought. It also reacts to salinity and the majority of hormonal treatments tested, but it does not respond to mechanical damage or treatments with methyl jasmonate. This is the difference with homologue enzymes present in other plant species, which have been related experimentally with abiotic stress. The accumulation patterns of transcripts in adult trees are compatible with a protector role against drought. The integration of our functional and phylogenetic studies suggests that the family has suffered a recent process of diversification and neofunctionalization, being the protection against drought their original role. Although the in vitro biochemistry activity of this kind of enzymes is already known, their natural substracts are essentially a mystery. By means of heterologous expression of Escherichia coli BL21(DE3) we have detected that the enzyme of poplar is able to oxidize L-DOPA to dopaquinone, being less active against other substrates. Additionally, we have proven its chloroplastic location with transitory transformation of protoplasts with YFP protein fusion. By means of getting transgenic plants of A. thaliana, we have demonstrated that the poplar enzyme increases notably the in vivo tolerance against the drought and salinity stresses. The phenotypic analysis of the transgenic lines, and the use of multiple methodologies, allowed us to test the complexity of the tolerance. This includes a major protection of the photosynthetic system, a very increased antioxidant capacity and the accumulation of osmoprotectant solutes as the proline. The metabolic analysis has allowed to associate the protein expression with the synthesis of a Flavan non described previously in A. thalaiana, linking the enzyme of poplar with the synthesis of phenylpropanoids. We have observed alterations in the hormonal levels that could underlie pleiotropic effects with applied interest, as a consistent increase of the size of the plant and the reduction of the growth cycle. The results of this thesis, in addition to provide novel data about the in vivo functionality of the oxidase family, demonstrate that the trees are interesting systems of study to characterize new strategies of tolerance against abiotic stress with applied potential.

Sola romani: morteros hidráulicos romanos en la Península Ibérica

En esta investigación se han analizado morteros de cal de cronología romana en el interior de la Península Ibérica. Para ello, se seleccionó una serie de muestras procedentes de diversos yacimientos, y de estructuras de carácter industrial. Estas muestras presentan aditivos cerámicos y conglomerantes de cal, como característica principal. Desde un principio el esfuerzo debía de centrarse en los fragmentos cerámicos presentes en los morteros. Para ello se documentaron varios morteros con aditivos cerámicos, a fin de conocer sus características básicas (componentes, distribución, micro-estratigrafía, granulometría, etc.). Por ello, y una vez tomadas las muestras, ésta fueron tratadas y procesadas para desarrollar una primera fase de estudio por medio de macroscopía. Se obtuvieron buenos resultados en cuanto a la caracterización visual de los morteros, localizando y documentando los fragmentos cerámicos. Durante la observación de dichos aditivos se observó que los fragmentos cerámicos tenían unas coronas o anillos de coloración que recorrían el borde, justo al contacto con la matriz de cal. Fueron seleccionados algunos fragmentos en los que eran más visibles dichos anillos. A fin de conocer la posible relación entre algunos yacimientos cercanos y la presencia de dicho anillo cromáticos se desarrolló una microscopía óptica polarizada, realizando láminas delgadas de las muestras y sobre los fragmentos cerámicos más determinantes, en concreto de una serie de enclaves del valle del Henares. Además de caracterizar microscópicamente los morteros, se observó que los anillos no eran un defecto visual ni una alteración física, producida tal vez por la extracción, la fase del corte o por la cocción de la cerámica. Tras analizar varios ejemplares se pudo apreciar que esos anillos eran habituales en todas las muestras y que se presentaban de diferente manera, es decir, que se observaban en granos cerámicos de diferentes características físicas (cochura, tamaño, situación con respecto del conglomerante, etc.). A fin de conocer los aspectos químicos de dicha alteración se seleccionó un grupo de muestras en las que los resultados macroscópicos habían sido muy claros, y en las que la microscopía óptica polarizada había determinado en los bordes, áreas adecuadas para otros análisis. Se realizó un mapeado o mapping de elementos químicos, a fin de saber qué podía estar sucediendo en esa interfaz entre el fragmento cerámico y la matriz de cal. Gracias a los resultados obtenidos se comprobó que existía una acumulación potencial de calcio tanto en el interior de los granos cerámicos como en el exterior, justo en la zona de contacto con el conglomerante, formando manchas que recorrían longitudinalmente el borde. Estos datos fueron muy útiles para llevar a cabo la siguiente fase de estudio, que permitiría conocer puntualmente qué estaba sucediendo químicamente en esa zona de contacto. Finalmente y con el objetivo de describir química y puntualmente este efecto en las adiciones cerámicas, se volvió a seleccionar una serie de muestras, escogidas esta vez en función de los granos cerámicos que cumpliesen unas variables estadísticas. Se eligieron granos con distintos tipos de cocciones, así como granos con tamaños diversos y granos con aditivo ceniciento en la matriz de cal, pensando que podrían ser las variables más útiles de interpretar de existir algún tipo de cambio químico entre la arcilla cocida y la matriz de cal. Tales variables se adaptaron a un sistema estadístico multi-varial y geométrico, con el objetivo de sintetizar los resultados y visualizar de forma óptima los datos en conjunto, como se ha comentado en varias ocasiones en este trabajo. Una vez seleccionados los granos por variables se procedió a realizar un análisis lineal y espectral semi-cuantitativo de SEM-EDX, con el que se caracterizaba químicamente una sección lineal del grano, desde la matriz de arcilla cocida de la cerámica hasta la matriz de cal –del interior al exterior del grano- pasando por el centro de la banda de reacción. Este análisis permitió determinar que se había producido una serie de cambios químicos porcentuales en los granos de cerámica. Dichos cambios se resumen en un incremento global de los porcentajes de calcio en el interior de las bandas de reacción de la cerámica, desde el borde mismo del anillo hasta el exterior. Así también se observaron picos porcentuales en el interfaz del fragmento cerámico con la matriz de cal, lo que confirmaba los resultados obtenidos por medio del mapping. Globalmente en todas las muestras se apreció un hombro en las gráficas de calcio a su paso por la zona de afección del anillo de reacción. Los restantes porcentajes de magnesio, silicio y aluminio se mantienen normales. En esta tesis se ha confirmado que dicho incremento global de calcio se acentúa en las muestras en donde no hay cenizas en la matriz de cal. Los casos correspondientes a estos granos sufren un incremento mayor que en el resto. La segunda variable que sufre un mayor incremento de calcio es la que corresponde a granos con buena cocción de la arcilla. Por lo tanto, parece que la tercera variable, la que corresponde con el tamaño del fragmento cerámico, no es decisiva. Por lo tanto, teniendo en cuenta la prueba visual de los anillos de reacción, y atendiendo a los resultados químicos, podríamos pensar que ese incremento de calcio en la banda de reacción de los fragmentos cerámicos se debió a una absorción de calcio en el interior de la arcilla cocida en la fase inmediatamente previa al fraguado, incluso durante el apagado de la cal. Es en este punto donde estaría la clave del cambio químico que se produce en esta interfaz, el calcio sílice-aluminato del que algunos autores ya han investigado. Esta absorción de calcio en el interior del grano no vendría sola, sino que generaría una costra de cal en el interfaz exterior de la cerámica, la cual ha sido observada químicamente por mapping y mineralógicamente por medio de microscopía óptica de polarización. La consecuencia de estos resultados es, primero, la mejora de nuestro conocimiento general acerca del factor hidráulico en los morteros. Asimismo se aprecia que la incorporación de materiales orgánicos como cenizas, puede alterar los porcentajes de calcio en el interior de los aditivos cerámicos, por lo que habría que sopesar, en trabajos futuros, si este tipo de material es adecuado o no en las mezclas destinadas a la restauración, así como seguir indagando en las propiedades de los morteros con la incorporación de aditivos orgánicos. Desde el punto de vista de la catalogación de los suelos industriales hidráulicos de época romana, además de mejorar la documentación incorporando micro-estratigrafías y granulometrías, la investigación de este material histórico constructivo mejora en cuanto a que se incorporan ensayos sencillos, que facilitan incluso la peritación de un estado de conservación por medio de una lupa binocular. Arqueológicamente hablando, es muy interesante correlacionar fábricas diferentes de estructuras situadas o bien en un mismo yacimiento, o bien en una misma área regional. Los estudios de caracterización y micro-estratigrafía no sólo aportan datos nuevos de cara a la restauración de morteros, sino que crean la posibilidad de generar patrones constructivos que sirvan de fósiles-guía para datar relativamente a unas estructuras o a otras. En lo referido a los resultados obtenidos en los diferentes complejos arqueológicos se ha observado una diferencia entre los morteros destinados a piletas y cubetas con respecto a los suelos industriales de uso indeterminado. La muestra correspondiente al yacimiento de Las Arenas no dispone de ninguna micro-estratigrafía, como sí por el contrario en las muestras obtenidas en Rotonda de Mejorada, Val de la Viña y La Magdalena. En estos enclaves las estructuras presentan grandes similitudes, con diferentes niveles constructivos empleando morteros de cal con áridos y gravas en las capas interiores, y áridos con adiciones cerámicas en las exteriores. En lo relativo a la granulometría las adiciones cerámicas de las muestras de Val de la Viña y La Magdalena presentan varias coincidencias en cuanto al tamaño de los granos y la distribución. Asimismo, de las muestras tomadas en La Magdalena, existe una gran diferencia entre las muestras MG1, MG2, MG3 y MG4 con respecto a las muestras MG5 y MG6, correspondientes éstas últimas a un mortero con fragmentos cerámicos de gran tamaño. Las estructuras EMG1 y EMG2, correspondientes a una cubeta y una pileta de La Magdalena, guardan similitud en lo referido a la micro-estratigrafía y a la granulometría. Se ha determinado que su función, así como su fabricación, debieron estar vinculadas a un mismo contexto cronocultural. Química y mineralógicamente, las muestras presentan características iguales, con presencia de un conglomerante de cal con áridos y aditivos cerámicos de diferentes cochuras. Destaca la muestra de Las Arenas, con un tamaño de los fragmentos cerámicos muy superior al resto, seguido de la estructura EMG3 de La Magdalena. Las muestras restantes de éste enclave, junto con las muestras recogidas en Val de la Viña y Rotonda de Mejorada presentan condiciones similares. En conclusión, los datos revelan que existían diferentes fábricas destinadas a distintas finalidades, y que las estructuras industriales empleaban aditivos cerámicos para la manipulación de productos con líquidos, de distintas densidades pero que requerían de cierto grado de hidraulicidad. ABSTRACT Lime Roman mortars from the Iberian Peninsula has been analyzed in this reesearch. A group of samples were selected from some sites and all the samples come from industrial structures. All this samples show ceramic additives. From the start, the effort was centered in the pieces of pottery that were found in the mortars. The samples were treated and processed to develop a first phase of the research using macroscopy. With this technique, great results were achieved in the characterization of mortars, the microstratigraphy and the location of the ceramic pieces. While observing these pieces, it was seen that the fragments of pottery had a ring bordering the piece. The pieces with the bigger and more vivid rings were chosen and they were analyzed by a polarized light microscope. The mortars were characterized microscopically and it also showed that the rings were not a physical alteration or a visual defect. After some more tests, the rings were a chemical change associated with the hydraulicity of the mortar. The best samples were selected and mappings of their chemical elements were performed in order to know what could be happening in the interface between the ceramic matrix fragment and lime. With the results obtained it was found that there was a potential; both calcium accumulation within the ceramic grains and outside, just in the area of contact with the binder, forming spots longitudinally along the edge. These data were very useful for carrying out the next phase of study, which would meet promptly what was happening chemically in the area of contact. Another group of samples were taken, and this time focused on ceramic grains that met a statistical variables. Grains were chosen with two types of cooking as well as grains with different sizes and grains with ash additive in the matrix of lime, thinking that might be the most logical to be some sort of chemical change between the baked clay and lime array variables . Such variables were adapted to a multi-varial and geometric statistical system in order to synthesize the results and optimally display the data together, as mentioned several times in this work. After selecting the variables grains proceeded to perform a linear and spectral analysis SEM-EDX. This analysis led to determine that the chemical changes were graduals. These changes are summarized in an increase in the percentages of calcium inside the reaction rim of ceramics, from the edge to the outer ring. So percentage increasing is also observed at the interface of the ceramic matrix fragment with lime, confirming the results obtained by the mapping. Overall in all samples can be seen a shoulder in graphic calcium through the area of the ring reaction condition. The remaining percentages of magnesium, silicon and aluminum are usual. We have promptly confirmed that the increase of calcium is accentuated in samples where there is no ash and lime matrix. Cases for these grains suffer a greater increase than the rest. The second variable suffering more calcium is increased corresponding to good cooking grains with clay. Therefore, it appears that the variable size of the fragment is not critical. Therefore, considering the visual tests to the rings and their response to chemical results, we might think that increasing calcium inside the ceramic fragments was due to an injection of calcium inside clay in the run-up to the setting phase. It is at this point that would be the key to the chemical change that occurs at this interface, silica-calcium aluminate some authors have already investigated. This injection of calcium into the grain does not come alone, but generate a lime crust on the outside interface of ceramics, which we tested for mapping is real in our samples. The consequence of these results is the improvement of our understanding of historical hydraulic factor in building materials, such as mortar. For example, knowing that the incorporation of organic materials such as ash powder, may be detrimental to the injection of calcium inside the ceramic additives. Archaeologically speaking, it's very interesting to correlate different factories or structures located on a single site, or in the same regional area. Characterization studies and microstratigraphy not only provide new information to help restore mortars, but create the possibility of generating constructive patterns that serve as guide fossils to determinate the age of the structures. With regard to the results obtained in different archaeological sites it has seen a difference between mortars of pools or sinks with respect to industrial floors of undetermined use. The sample of the site of Las Arenas does not have any micro-stratigraphy, as if instead in the samples obtained in Rotonda de Mejorada, Val de la Viña and La Magdalena sites. In these settlements the structures are really similar, with different construction levels using lime mortars with aggregates and gravel in the inner layers, and ceramic aggregates as external additions. With regard to the grain size of the ceramic additions Val de la Viña and La Magdalena samples has several coincidences about the size of grains and distribution. Also, samples taken at La Magdalena, there is a difference between the MG1, MG2, MG3 and MG4 samples and the MG5 and MG6 samples, so the last corresponding to a mortar samples with larger ceramic fragments. The EMG1 and EMG2 structures, corresponding to a bucket and a pool of La Magdalena settlement, have similarities with regard to micro-stratigraphy and grain size. It has been determined that the function and manufacturing must be linked with a same chronocultural context.

Regulation of T cell receptor (TCR) β gene expression by CD3 complex signaling in immature thymocytes: Implications for TCRβ allelic exclusion

During αβ thymocyte development, clonotype-independent CD3 complexes are expressed at the cell surface before the pre-T cell receptor (TCR). Signaling through clonotype-independent CD3 complexes is required for expression of rearranged TCRβ genes. On expression of a TCRβ polypeptide chain, the pre-TCR is assembled, and TCRβ locus allelic exclusion is established. We investigated the putative contribution of clonotype-independent CD3 complex signaling to TCRβ locus allelic exclusion in mice single-deficient or double-deficient for CD3ζ/η and/or p56lck. These mice display defects in the expression of endogenous TCRβ genes in immature thymocytes, proportional to the severity of CD3 complex malfunction. Exclusion of endogenous TCRβ VDJ (variable, diversity, joining) rearrangements by a functional TCRβ transgene was severely compromised in the single-deficient and double-deficient mutant mice. In contrast to wild-type mice, most of the CD25+ double-negative (DN) thymocytes of the mutant mice failed to express the TCRβ transgene, suggesting defective expression of the TCRβ transgene similar to endogenous TCRβ genes. In the mutant mice, a proportion of CD25+ DN thymocytes that failed to express the transgene expressed endogenous TCRβ polypeptide chains. Many double-positive cells of the mutant mice coexpressed endogenous and transgenic TCRβ chains or more than one endogenous TCRβ chain. The data suggest that signaling through clonotype-independent CD3 complexes may contribute to allelic exclusion of the TCRβ locus by inducing the expression of rearranged TCRβ genes in CD25+ DN thymocytes.

Effect of mutating the regulatory phosphoserine and conserved threonine on the activity of the expressed catalytic domain of Acanthamoeba myosin I heavy chain kinase

Phosphorylation of Ser-627 is both necessary and sufficient for full activity of the expressed 35-kDa catalytic domain of myosin I heavy chain kinase (MIHCK). Ser-627 lies in the variable loop between highly conserved residues DFG and APE at a position at which a phosphorylated Ser/Thr also occurs in many other Ser/Thr protein kinases. The variable loop of MIHCK contains two other hydroxyamino acids: Thr-631, which is conserved in almost all Ser/Thr kinases, and Thr-632, which is not conserved. We determined the effects on the kinase activity of the expressed catalytic domain of mutating Ser-627, Thr-631, and Thr-632 individually to Ala, Asp, and Glu. The S627A mutant was substantially less active than wild type (wt), with a lower kcat and higher Km for both peptide substrate and ATP, but was more active than unphosphorylated wt. The S627D and S627E mutants were also less active than phosphorylated wt, i.e., acidic amino acids cannot substitute for phospho-Ser-627. The activity of the T631A mutant was as low as that of the S627A mutant, whereas the T632A mutant was as active as phosphorylated wt, indicating that highly conserved Thr-631, although not phosphorylated, is essential for catalytic activity. Asp and Glu substitutions for Thr-631 and Thr-632 were inhibitory to various degrees. Molecular modeling indicated that Thr-631 can hydrogen bond with conserved residue Asp-591 in the catalytic loop and that similar interactions are possible for other kinases whose activities also are regulated by phosphorylation in the variable loop. Thus, this conserved Thr residue may be essential for the activities of other Ser/Thr protein kinases as well as for the activity of MIHCK.

Force-mediated kinetics of single P-selectin/ligand complexes observed by atomic force microscopy

Leukocytes roll along the endothelium of postcapillary venules in response to inflammatory signals. Rolling under the hydrodynamic drag forces of blood flow is mediated by the interaction between selectins and their ligands across the leukocyte and endothelial cell surfaces. Here we present force-spectroscopy experiments on single complexes of P-selectin and P-selectin glycoprotein ligand-1 by atomic force microscopy to determine the intrinsic molecular properties of this dynamic adhesion process. By modeling intermolecular and intramolecular forces as well as the adhesion probability in atomic force microscopy experiments we gain information on rupture forces, elasticity, and kinetics of the P-selectin/P-selectin glycoprotein ligand-1 interaction. The complexes are able to withstand forces up to 165 pN and show a chain-like elasticity with a molecular spring constant of 5.3 pN nm−1 and a persistence length of 0.35 nm. The dissociation constant (off-rate) varies over three orders of magnitude from 0.02 s−1 under zero force up to 15 s−1 under external applied forces. Rupture force and lifetime of the complexes are not constant, but directly depend on the applied force per unit time, which is a product of the intrinsic molecular elasticity and the external pulling velocity. The high strength of binding combined with force-dependent rate constants and high molecular elasticity are tailored to support physiological leukocyte rolling.

Major histocompatibility complex class I-restricted T cells are required for all but the end stages of diabetes development in nonobese diabetic mice and use a prevalent T cell receptor α chain gene rearrangement

Nonobese diabetic (NOD) mice develop insulin-dependent diabetes mellitus due to autoimmune T lymphocyte-mediated destruction of pancreatic β cells. Although both major histocompatibility complex class I-restricted CD8+ and class II-restricted CD4+ T cell subsets are required, the specific role each subset plays in the pathogenic process is still unclear. Here we show that class I-dependent T cells are required for all but the terminal stages of autoimmune diabetes development. To characterize the diabetogenic CD8+ T cells responsible, we isolated and propagated in vitro CD8+ T cells from the earliest insulitic lesions of NOD mice. They were cytotoxic to NOD islet cells, restricted to H-2Kd, and showed a diverse T cell receptor β chain repertoire. In contrast, their α chain repertoire was more restricted, with a recurrent amino acid sequence motif in the complementarity-determining region 3 loop and a prevalence of Vα17 family members frequently joined to the Jα42 gene segment. These results suggest that a number of the CD8+ T cells participating in the initial phase of autoimmune β cell destruction recognize a common structural component of Kd/peptide complexes on pancreatic β cells, possibly a single peptide.

Exploring the origins of topological frustration: Design of a minimally frustrated model of fragment B of protein A

Topological frustration in an energetically unfrustrated off-lattice model of the helical protein fragment B of protein A from Staphylococcus aureus was investigated. This Gō-type model exhibited thermodynamic and kinetic signatures of a well-designed two-state folder with concurrent collapse and folding transitions and single exponential kinetics at the transition temperature. Topological frustration is determined in the absence of energetic frustration by the distribution of Fersht φ values. Topologically unfrustrated systems present a unimodal distribution sharply peaked at intermediate φ, whereas highly frustrated systems display a bimodal distribution peaked at low and high φ values. The distribution of φ values in protein A was determined both thermodynamically and kinetically. Both methods yielded a unimodal distribution centered at φ = 0.3 with tails extending to low and high φ values, indicating the presence of a small amount of topological frustration. The contacts with high φ values were located in the turn regions between helices I and II and II and III, intimating that these hairpins are in large part required in the transition state. Our results are in good agreement with all-atom simulations of protein A, as well as lattice simulations of a three- letter code 27-mer (which can be compared with a 60-residue helical protein). The relatively broad unimodal distribution of φ values obtained from the all-atom simulations and that from the minimalist model for the same native fold suggest that the structure of the transition state ensemble is determined mostly by the protein topology and not energetic frustration.

Coexpression of factor VIII heavy and light chain adeno-associated viral vectors produces biologically active protein

We are interested in using recombinant adeno-associated viral vectors in the treatment of hemophilia A. Because of the size constraints of recombinant adeno-associated viral vectors, we delivered the heavy and light chains of the human factor 8 (hFVIII) cDNA independently by using two separate vectors. Recombinant AAV vectors were constructed that utilized the human elongation factor 1α promoter, a human growth factor polyadenylation signal, and the cDNA sequences encoding either the heavy or light chain of hFVIII. Portal vein injections of each vector alone, a combination of both vectors, or a hFIX control vector were performed in C57BL/6 mice. An ELISA specific for the light chain of hFVIII demonstrated very high levels (2–10 μg/ml) of protein expression in animals injected with the light chain vector alone or with both vectors. We utilized a chromogenic assay in combination with an antibody specific to hFVIII to determine the amount of biologically active hFVIII in mouse plasma. In animals injected with both the heavy and light chain vectors, greater than physiological levels (200–400 ng/ml) of biologically active hFVIII were produced. This suggests that coexpression of the heavy and light chains of hFVIII may be a feasible approach for treatment of hemophilia A.

Demonstration of a novel circulating anti-prostacyclin receptor antibody

Although coronary artery disease (CAD) is appreciated to be accelerated in patients with chronic spinal cord injury (SCI), the underlying mechanism of CAD in SCI remains obscure. We have recently shown that platelets from subjects with SCI develop resistance to the inhibitory effect of prostacyclin (PGI2) on the platelet stimulation of thrombin generation. The loss of the inhibitory effect was due to the loss of high-affinity prostanoid receptors, which may contribute to atherogenesis in SCI. Incubation of normal, non-SCI platelets in SCI plasma (n = 12) also resulted in the loss of high-affinity binding of PGI2 (Kd1 = 9.1 ± 2.0 nM; n1 = 170 ± 32 sites per cell vs. Kd1 = 7.2 ± 1.1 nM; n1 = 23 ± 8 sites per cell), with no significant change in the low-affinity receptors (Kd2 = 1.9 ± 0.1 μM; n2 = 1,832 ± 232 sites per cell vs. Kd2 = 1.6 ± 0.1 μM; n2 = 1,740 ± 161 sites per cell) as determined by Scatchard analysis of the binding of [3H]PGE1. The loss of high-affinity PGI2 binding led to the failure of PGI2 to inhibit the platelet-stimulated thrombin generation. The increase of cellular cyclic AMP level, mediated through the binding of PGI2 to low-affinity receptors in platelets, was unaffected in SCI platelets. PAGE and immunoblot of SCI plasma showed the presence of an IgG band, which specifically blocked the binding of [3H]PGE1 to the high-affinity PGI2 receptors of normal platelets. PAGE of the reduced IgG band, the amino acid sequence of the novel band as a heavy chain of IgG that inhibits the binding of [3H]PGE1 to the high-affinity platelet PGI2 receptor, demonstrates that the specific recognition and inhibition of high-affinity PGI2 binding to platelets was due to an anti-prostacyclin receptor antibody present in SCI plasma.

Hodgkin and Reed–Sternberg cells in lymphocyte predominant Hodgkin disease represent clonal populations of germinal center-derived tumor B cells

Among the four subtypes of Hodgkin disease (HD), lymphocyte-predominant (LP) HD is now generally considered as a separate entity. The B cell nature of the typical Hodgkin and Reed–Sternberg (HRS) cells and their variants (L and H, lymphocytic and histiocytic cells) in LP HD has long been suspected, but the question of whether these cells represent a true tumor clone is unclear. We previously demonstrated clonal Ig gene rearrangements in one case of LP HD. In the present study, five cases of LP HD were analyzed by micromanipulation of single HRS cells from frozen tissue sections and DNA amplification of rearranged Ig heavy chain genes from those cells. Clonal V gene rearrangements harboring somatic mutations were detected in each case. In three cases ongoing somatic mutation was evident. This shows that HRS cells in LP HD are a clonal tumor population derived from germinal center B cells. The pattern of somatic mutation indicates that HRS cells in LP HD are selected for antibody expression. This, and the presence of ongoing mutation discriminates LP from classical HD.

Solution structure of a 142-residue recombinant prion protein corresponding to the infectious fragment of the scrapie isoform

The scrapie prion protein (PrPSc) is the major, and possibly the only, component of the infectious prion; it is generated from the cellular isoform (PrPC) by a conformational change. N-terminal truncation of PrPSc by limited proteolysis produces a protein of ≈142 residues designated PrP 27–30, which retains infectivity. A recombinant protein (rPrP) corresponding to Syrian hamster PrP 27–30 was expressed in Escherichia coli and purified. After refolding rPrP into an α-helical form resembling PrPC, the structure was solved by multidimensional heteronuclear NMR, revealing many structural features of rPrP that were not found in two shorter PrP fragments studied previously. Extensive side-chain interactions for residues 113–125 characterize a hydrophobic cluster, which packs against an irregular β-sheet, whereas residues 90–112 exhibit little defined structure. Although identifiable secondary structure is largely lacking in the N terminus of rPrP, paradoxically this N terminus increases the amount of secondary structure in the remainder of rPrP. The surface of a long helix (residues 200–227) and a structured loop (residues 165–171) form a discontinuous epitope for binding of a protein that facilitates PrPSc formation. Polymorphic residues within this epitope seem to modulate susceptibility of sheep and humans to prion disease. Conformational heterogeneity of rPrP at the N terminus may be key to the transformation of PrPC into PrPSc, whereas the discontinuous epitope near the C terminus controls this transition.

The α chain of laminin-1 is independently secreted and drives secretion of its β- and γ-chain partners

A mammalian recombinant strategy was established to dissect rules of basement membrane laminin assembly and secretion. The α-, β-, and γ-chain subunits of laminin-1 were expressed in all combinations, transiently and/or stably, in a near-null background. In the absence of its normal partners, the α chain was secreted as intact protein and protein that had been cleaved in the coiled-coil domain. In contrast, the β and γ chains, expressed separately or together, remained intracellular with formation of ββ or βγ, but not γγ, disulfide-linked dimers. Secretion of the β and γ chains required simultaneous expression of all three chains and their assembly into αβγ heterotrimers. Epitope-tagged recombinant α subunit and recombinant laminin were affinity-purified from the conditioned medium of αγ and αβγ clones. Rotary-shadow electron microscopy revealed that the free α subunit is a linear structure containing N-terminal and included globules with a foreshortened long arm, while the trimeric species has the typical four-arm morphology of native laminin. We conclude that the α chain can be delivered to the extracellular environment as a single subunit, whereas the β and γ chains cannot, and that the α chain drives the secretion of the trimeric molecule. Such an α-chain-dependent mechanism could allow for the regulation of laminin export into a nascent basement membrane, and might serve an important role in controlling basement membrane formation.

Functional cleavage of the common cytokine receptor γ chain (γc) by calpain

The small subunit of calpain, a calcium-dependent cysteine protease, was found to interact with the cytoplasmic domain of the common cytokine receptor γ chain (γc) in a yeast two-hybrid interaction trap assay. This interaction was functional as demonstrated by the ability of calpain to cleave in vitro-translated wild-type γc, but not γc containing a mutation in the PEST (proline, glutamate, serine, and threonine) sequence in its cytoplasmic domain, as well as by the ability of endogenous calpain to mediate cleavage of γc in a calcium-dependent fashion. In T cell receptor-stimulated murine thymocytes, calpain inhibitors decreased cleavage of γc. Moreover, in single positive CD4+ thymocytes, not only did a calpain inhibitor augment CD3-induced proliferation, but antibodies to γc blocked this effect. Finally, treatment of cells with ionomycin could inhibit interleukin 2-induced STAT protein activation, but this inhibition could be reversed by calpain inhibitors. Together, these data suggest that calpain-mediated cleavage of γc represents a mechanism by which γc-dependent signaling can be controlled.

Defect in IgV gene somatic hypermutation in Common Variable Immuno-Deficiency syndrome

Common Variable Immuno-Deficiency (CVID) is the most common symptomatic primary antibody-deficiency syndrome, but the basic immunologic defects underlying this syndrome are not well defined. We report here that among eight patients studied (six CVID and two hypogammaglobulinemic patients with recurrent infections), there is in two CVID patients a dramatic reduction in Ig V gene somatic hypermutation with 40–75% of IgG transcripts totally devoid of mutations in the circulating memory B cell compartment. Functional assays of the T cell compartment point to an intrinsic B cell defect in the process of antibody affinity maturation in these two cases.

Sequence dependent hypermutation of the immunoglobulin heavy chain in cultured B cells

The variable (V) regions of immunoglobulin heavy and light chains undergo high rates of somatic mutation during the immune response. Although point mutations accumulate throughout the V regions and their immediate flanking sequences, analysis of large numbers of mutations that have arisen in vivo reveal that the triplet AGC appears to be most susceptible to mutation. We have stably transfected B cell lines with γ2a heavy chain constructs containing TAG nonsense codons in their V regions that are part of either a putative (T)AGC hot spot or a (T)AGA non-hot spot motif. Using an ELISA spot assay to detect revertants and fluctuation analysis to determine rates of mutation, the rate of reversion of the TAG nonsense codon has been determined for different motifs in different parts of the V region. In the NSO plasma cell line, the (T)AGC hot spot motif mutates at rates of ≈6 × 10−4/bp per generation and ≈3 × 10−5/bp per generation at residues 38 and 94 in the V region. At each of these locations, the (T)AGC hot spot motif is 20–30 times more likely to undergo mutation than the (T)AGA non-hot spot motif. Moreover, the AGA non-hot spot motif mutates at as high a rate as the hot spot motif when it is located adjacent to hot spot motifs, suggesting that more extended sequences influence susceptibility to mutation.